Expression Validation by qRT-PCR
To validate and check the reliability of RNA-Seq data, qRT-PCR analysis was performed. Twelve stress induced genes were selected for experimental validation by qRT-PCR. From each biological replicate, cDNA was synthesized using the HiScriptII-QRT-superMix through qPCR+ gDNA wiper (Vazyme, Nanjing, China) in accordance with manufacturer’s instruction. ABI 7500 Real-Time PCR System (Applied Biosystems, USA) was used for qRT-PCR analysis. Each reaction comprises of 10µl 2×SYBR Green Master Mix (Applied Biosystems, USA), 2.0µl cDNA, and 400 nM primer (forward, reverse) upto final volume of 20µl. The PCR conditions were 95˚C for 3 min followed by 40 cycles of 95˚C for 10 s, 60˚C for 30 s and 65˚C for 5s. Moreover, expression level were normalized by using actin gene (GmActin11 , as an internal control) and Ct method (Livak and Schmittgen, 2001). For each sample, qRT-PCR reaction was repeated in two replicates, and three technical repeats of each biological replicate. The PRIMER5.0 software was used to design primers, and all primers are listed in Supplementary Table S1.